The term "Escherichia coli" as used herein, refers to bacteria classified as such in Bergey's Manual of Systematic Bacteriology (N. R. Krieg [ed.1, 1984, pp408-423, Williams & Wilkins). Detection of Escherichia coli (E. coli) is important in various medical and public health contexts. Escherichia coli (E. coli) was discovered to be ubiquitous in fecal material nearly a century ago. Thus, foods are tested for E. coli as the indicator organism for fecal contamination. Generally, the presence of E. coli in food and water is used as a measure of sanitary conditions. E. coli infection itself also can cause a variety of symptoms ranging from mild to severe gastroenteritis. A large variety of food and environmental samples are potential sources of human E. coli infection but routine screening is both time consuming and difficult.
It is, therefore, an aspect of the present invention to provide a novel assay system capable of rapidly detecting E. coli and which is generally applicable to environmental, food or clinical samples. The probes of the present invention also detect all Shigella species and strains tested to date (Table 2). Shigella, as used herein, refers to bacteria classified as such in Bergey's Manual of Systematic Bacteriology (ibid., pp. 423-427). Shigella is primarily a pathogen of man and other primates, and is a causative agent of bacillary dysentery. Members of the genus Shigella are extremely closely related to members of the genus Escherichia and exhibit considerable overlap in genetic, biochemical and pathogenic characteristics with members of the latter genus. Although Shigella is only rarely isolated from food samples or from fecal material of normal healthy humans, its presence in a test sample would clearly also be an indication of fecal contamination.
It is another aspect of the present invention to provide a diagnostic assay for the detection of E. coli plus Shigella in a test sample.
Pursuant to a standard laboratory method and a method recommended by the FDA (FDA/BAM Bacteriological Analytical Manual, Chapters 5 and 6, 6th Edition, 1984, Supplement 9/87', Association of Offical Analytical Chemists), the presence of E. coli has been traditionally detected by culturing an appropriately prepared sample on microbiological media under conditions favorable for growth of these organisms. The resulting colonies are then typically examined for morphological and biochemical characteristics, a process that generally is initiated 48 hours after acquisition of the sample and disadvantageously takes between four to six days to complete.
A recent, more rapid method for detection of E. coli, developed by Feng and Harmant (Appl. and Environ. Microbiol., 1982, 43:1320-1329), uses 4-methylumbelliferyl-.beta.-D-glucuronide (MUG). The MUG assay is a fluorogenic test for the enzyme .beta.-glucuronidase. According to Kilian and Bulow (Acta Path. Microbiol. Scand., Sect. B, 1976, 84:245-251), 97% of E. coli strains possess .beta.-glucuronidase. The basic premise is that the rapid confirmation of E. coli is possible by incorporating MUG into a suitable culture broth, such as LST or EC. After inoculation of the broth with a test sample, fluorescence of 4-methylumbelliferone is produced from hydrolysis of MUG if .beta.-glucuronidase is present, and can be determined by examining the sample under long-wave ultraviolet light (366 nanometers) after 24-48 hours of incubation. In the commonly used format of this test, a positive E. coli sample shows: (1) gas production in a Durham tube, and (2) fluorescence of the broth upon illumination with ultraviolet light. However, as demonstrated by the results shown in Table 2, the MUG test yields ambiguous patterns of gas production and fluorescence for many E. coli strains. Particularly striking is the apparent lack of fluorescence (and, by inference, .beta.-glucuronidase) in all tested strains of serotype .alpha. 0:157 H7 enteropathogenic E. coli.
It is yet another aspect of the present invention to avoid the disadvantages associated with these techniques and to employ nucleic acid probes to detect E. coli and Shigella.
It is yet another aspect of the present invention to provide probes which can hybridize to target regions which can be rendered accessible to the probes under normal assay conditions.
While Kohne et al. (1968) Biophysical Journal 8:1104-1118 discuss one method for preparing probes to rRNA sequences they do not provide the teaching necessary to make nor can they predict the existence of these probes for the specific detection of E. coli and E. coli/Shigella.
Pace and Campbell (1971) Journal of Bacteriology 107:543-547 discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin, and Woese (1972) Journal of Molecular Evolution 1:173-184 discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships.
Fox, Pechman, and Woese (1977) International Journal of Systematic Bacteriology 27:44-57 discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to E. coli and Shigella.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, at least in E. coli, are referred to as 5S, 16S, and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by sedimentation rate. In actuality, however, they vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein.
Hybridization is traditionally understood as the process by which, under predetermined reaction conditions, two partially or completely complementary single-stranded nucleic acids are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds. The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types of concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will largely dictate the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art. In general, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially) to target nucleic acid sequences.
A target nucleic acid is one to which a particular probe is capable of preferentially hybridizing.
Still other useful definitions are given as their first use arises in the following text. All references cited herein are fully incorporated by reference.